Novel aliphatic compound, method of synthesis, and method of utilization

ABSTRACT

The present invention relates to aliphatic compounds of the formula I, or stereoisomers thereof, or their pharmaceutically acceptable salts:  
                 
 
     wherein A represents an optionally substituted CH 3 C n H (2n−2m) — (wherein n denotes an integer of 4 to 22, and m represents an unsaturation number which is an integer of 0 to 7), 1 represents an integer of 0 to 10, s represents 0 or 1, provided that when s is 0, p+q=4 or 5, but when s is 1, p+q=3 or 4, and in each case, either p or q is an integer of 1 or more, R represents an alkyl group having 1 to 10 carbon atoms which may be straight-chain or branched-chain, and R A  represents hydrogen or an alkyl group having 1 to 10 carbon atoms which may be straight-chain or branched-chain, and their use in suppression of platelet aggregation, in suppression of inflammation, and in prevention and treatment of circulatory diseases.

TECHNICAL FIELD

[0001] This invention relates to novel aliphatic compounds,pharmaceutical compositions containing them, and their use in thesuppression of platelet aggregation, the suppression of inflammation,and the prevention and treatment of circulatory diseases.

BACKGROUND ART

[0002] When platelets in the blood contact subendothelial tissue aftervascular endothelial cells are injured and peeled, they adhere theretoand cause an aggregation reaction. This reaction brings about thrombusformation, causing vascular disorders including thrombosis.

[0003] In the prevention and treatment of such diseases, therefore, itis important to elucidate platelet functions and consider how plateletaggregation should be suppressed.

[0004] In connection with adhesion and aggregation functions among theplatelet functions, the following theory is currently held: When astimulant, such as collagen, arachidonic acid, ADP, thrombin, serotoninor epinephrine, stimulates corresponding receptors on the plateletmembrane, the glycoprotein conjugate GPIIb-IIIa on the membrane becomescapable of binding to fibrinogen in the blood via the stimulusconducting system. As a result, platelets are mutually crosslinked andaggregated.

[0005] Much still remains unknown about the actions of the abovesubstances working as stimulants. However, it is speculated that stimulifrom various stimulants including collagen activate phospholipase A2 toproduce arachidonic acid from phospholipid, and the resultingarachidonic acid is metabolized into prostaglandins (PG)G₂ and PGH₂ bycyclooxygenase (COX), and further into thromboxane (TX)A₂.

[0006] Also, the actions of the above stimulants are different. Stimulifrom the stimulants, other than epinephrine and collagen, to plateletscause influx of Ca ions from outside of cells, and mobilize Ca ions fromCa storage granules, thereby raising intracellular Ca ion concentration.This causes the structural change of GPIIb-IIIa and contraction ofcontractile protein, arousing platelet aggregation and releasereactions. With collagen, such reactions have not been observed.

[0007] In terms of the mechanism of exhibition of such plateletfunctions, antiplatelet drugs currently developed are classified intothose acting on stimulus receptors, those acting on the stimulusconducting system (PG metabolism system inhibitors, those involved incAMP metabolism), and those acting on GPIIb-IIIa.

[0008] GPIIb-IIIa receptor antagonists inhibit the terminal point of theaforementioned platelet reaction, and thus inhibit every plateletreaction, regardless of the cause of the platelet reaction. On the otherhand, the potency of conventional GPIIb/IIIa receptor antagonists issuch that its effective dose in single dose treatment is about 0.1 to 1mg/kg by the intravenous route. Thus, this potency cannot be said to besufficiently high.

[0009] Hence, antiplatelet drugs, which suppress platelet aggregationpotently, are desired.

DISCLOSURE OF THE INVENTION

[0010] We, the present inventors, conducted in-depth studies in thelight of the above facts. As a result, we newly discovered compoundsrepresented by the general formula I shown below, or theirstereoisomers, and have found that these compounds (hereinafter,referred to as “compounds of the present invention” including theirstereoisomers) show a much more potent action of suppressing plateletaggregation than that of conventional GPIIb/IIIa receptor antagonists,and further exhibit an anti-inflammatory action. The present inventionis based on this finding, and its object is to provide novel aliphaticcompounds, a method for their production, and pharmaceuticals comprisingthem.

[0011] The present invention relates to aliphatic compounds of thegeneral formula I, or stereoisomers thereof, or their pharmaceuticallyacceptable salts:

[0012] wherein

[0013] A represents an optionally substituted CH₃C_(n)H_((2n−2m))— wheren denotes an integer of 4 to 22, and m represents an unsaturation numberwhich is an integer of 0 to 7,

[0014] l represents an integer of 0 to 10,

[0015] s represents 0 or 1, provided that when s is 0, p+q 4 or 5, butwhen s is 1, p+q=3 or 4, and in each case, either p or q is an integerof 1 or more,

[0016] R represents an alkyl group having 1 to 10 carbon atoms which maybe straight-chain or branched-chain, and

[0017] R^(A) represents hydrogen or an alkyl group having 1 to 10 carbonatoms which may be straight-chain or branched-chain.

[0018] (In connection with the positions of the unsaturated bonds ofCH₃C_(n)H_((2n−2m))— in the definition of A in the formula I, theposition of “C” of the amide bond NHCO is taken as 1, and the adjacentcarbons are sequentially numbered 2, 3, 4 . . . to show the positionsfor use in the explanation offered below.)

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a graph showing the action, by the compounds of thepresent invention (Compounds 1 to 9), of suppressing plateletaggregation in vitro dose-dependently as does indomethacin.

[0020]FIG. 2 is a graph showing changes over time in the action, by thecompound of the present invention (Compound 2), of suppressing plateletaggregation ex vivo.

[0021]FIG. 3 is a graph showing the action, by the compound of thepresent invention (Compound 2), of suppressing platelet aggregation exvivo dose-dependently.

[0022]FIG. 4 is a graph showing the action, by the compound of thepresent invention (Compound 5), of suppressing platelet aggregation exvivo.

[0023]FIG. 5 is a graph showing the action, by the compound of thepresent invention (Compound 2) absorbed orally, of suppressing plateletaggregation.

[0024]FIG. 6 is a graph showing that in the case of administration ofthe compound of the present invention after leg necrosis induction, thenecrosis score is improved dose-dependently in a peripheral circulatorydisorder model.

[0025]FIG. 7 is a graph showing that in the case of administration ofthe compound of the present invention before leg necrosis induction, thenecrosis score is improved dose-dependently in a peripheral circulatorydisorder model.

[0026]FIG. 8 is a graph showing that when an O/W emulsion of thecompound of the present invention is administered before leg necrosisinduction, the necrosis score is improved dose-dependently in aperipheral circulatory disorder model.

[0027]FIG. 9 is a graph showing that the compounds of the presentinvention (Compounds 1 and 2) act to suppress the migration ofneutrophils into vascular endothelial cells.

[0028]FIG. 10 is a graph showing that the compound of the presentinvention (Compound 2) ameliorates cerebral infarction in a middlecerebral artery obstruction model.

[0029]FIG. 11 is a graph showing that an O/W emulsion of the compound ofthe present invention suppresses vascular tunica intima thickeningdose-dependently in a post-PTCA restenosis model.

[0030]FIG. 12 is a graph showing the action, by the compound of thepresent invention (Compound 2), of suppressing vascular smooth musclecell proliferation dose-dependently.

BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The definitions in the formula I for the compounds of the presentinvention will be described.

[0032] Concrete examples of the “alkyl group having 1 to 10 carbon atomswhich may be straight-chain or branched-chain” are alkyl groups such asa methyl group, an ethyl group, an n-propyl group, an isopropyl group,an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butylgroup, an n-pentyl group, a tert-amyl group, a 3-methylbutyl group, aneopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group,an n-nonyl group, and an n-decyl group.

[0033] The term “optionally substituted CH₃C_(n)H_((2n−2m))—” refers toCH₃C_(n)H_((2n−2m))— may having any substituent.

[0034] Examples of the substituent include a hydroxyl group, a halogenatom, an alkyl group having 1 to 10 carbon atoms which may bestraight-chain or branched-chain, a cycloalkyl group having 3 to 7carbon atoms, and an aryl group.

[0035] Concrete examples of the “cycloalkyl group having 3 to 7 carbonatoms” include a cyclopropyl group, a cyclobutyl group, a cyclopentylgroup, a cyclohexyl group, and a cycloheptyl group.

[0036] Concrete examples of the “aryl group” include a phenyl group,etc.

[0037] Concrete examples of the “alkyl group having 1 to 10 carbon atomswhich may be straight-chain or branched-chain” are as described above.

[0038] Preferred embodiments of the compounds of the general formula Iaccording to the present invention are mentioned as follows:

[0039] Preferably, p and q are such that p=q=2.

[0040] The present invention provides the compounds of the generalformula I which are compounds of the following general formula II (thesecompounds correspond to the compounds of the general formula I whereins=0 and p=q=2):

[0041] General Formula II

[0042] wherein R, R^(A), A, and l have the same meanings as the meaningsof the symbols in the formula I.

[0043] In the above general formula II, the preferred substitutionposition of A-CONH—(CH₂)_(l)— is at the carbon adjacent to N—R.

[0044] The present invention provides the compounds of the generalformula I which are compounds of the following general formula III(these compounds correspond to the compounds of the general formula Iwhere s=1 and p=q=2):

[0045] General Formula III

[0046] where R, R^(A), A, and l have the same meanings as the meaningsof the symbols in the formula I.

[0047] In the above general formula III, the preferred substitutionposition of A-CONH—(CH₂)_(l)— is at the nitrogen atom of the piperazinering.

[0048] In the compounds of the general formulas I to III according tothe present invention, the preferred embodiments are as follows:

[0049] R is preferably methyl, ethyl, propyl, isopropyl or butyl, morepreferably methyl or ethyl, and most preferably a methyl group.

[0050] R^(A) is preferably hydrogen, but when R^(A) is an alkyl group,it preferably has 1 to 6 carbon atoms, and more preferably it has 1 to 4carbon atoms. The position of substitution by R^(A) in the ring ispreferably a position which is not adjacent to N—R.

[0051] l is preferably an integer of 0 to 3.

[0052] n is preferably an integer of 6 to 22, and more preferably aninteger of 14 to 22.

[0053] m is preferably an integer of 1 to 7, and more preferably aninteger of 2 to 6.

[0054] Preferred examples of A are derived from, but not limited to,docosahexaenoic acid (n=20, m=6) or eicosapentaenoic acid (n=18, m=5).

[0055] In connection with the positions of the unsaturated bonds ofCH₃C_(n)H_((2n−2m))— in the definition of A, they are preferably, thepositions 9, 12 and 15 if n=16, the positions 5, 8, 11, 14 and 17 ifn=18, and the positions 4, 7, 10, 13, 16 and 19 if n=20.

[0056] The optional substituent of A is preferably that which does notaffect the solubility of the compounds of the formula I. If thesubstituent is alkyl, it is preferably an alkyl group having a lowmolecular weight, for example, an alkyl group having 1 to 4 carbon atom,and more preferably a methyl group. The preferred substitution positionis at a position which is not in proximity to the amide bond. Forexample, the position is the position 3 to 23, more preferably theposition 3 to 20.

[0057] Preferred examples of A having the substituent, if they arederivatives having the substituent OH, include hydroxylated derivativesof docosahexaenoic acid (DHA), or hydroxylated derivatives ofeicosapentaenoic acid (EPA), and more preferably hydroxylatedderivatives of docosahexanoic acid (DHA). The steric configuration ofthe hydroxylated derivatives may be (R)-configuration or(S)-configuration.

[0058] Examples of the hydroxylated derivatives of docosahexaenoic acid(DHA) include, but not limited to, 4-OH-DHA, 10-OH-DHA, 11-OH-DHA,14-OH-DHA, 8-OH-DHA and 17-OH-DHA.

[0059] Examples of the hydroxylated derivatives of eicosapentaenoic acid(EPA) include 12-OH-EPA, which is not limitative. (For the abovehydroxylated derivatives, see J. W. Karanian et al., The Journal ofPharmacology and Experimental Therapeutics (1994) 270, 1105-1109.)

[0060] As the preferred compounds of the present invention, thefollowing compounds, their optical isomers, or their pharmaceuticallyacceptable salts are listed:

[0061](4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]docosahexaenoamide;

[0062](4Z,7Z,10Z,13Z,16Z,19Z)-N-(4-methylpiperazin-1-yl)docosahexaenoamide;

[0063] N-[2-(1-methylpyrrolidin-2-yl)ethyl]caprylamide;

[0064] N-[2-(1-methylpyrrolidin-2-yl)ethyl]myristamide;

[0065] 9Z-N-[2-(1-methylpyrrolidin-2-yl)ethyl]oleamide;

[0066] (9Z,12Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]linoleamide;

[0067] (9Z,12Z,15Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]linolenamide;

[0068](5Z,8Z,11Z,14Z,17Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]eicosapentaenoamide;

[0069](4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)methyl]docosahexaenoamide;and

[0070](4Z,7Z,10Z,13Z,16Z,19Z)-N-[3-(1-methylpyrrolidin-2-yl)propyl]docosahexaenoamide.

[0071] Further preferred compounds as the compounds of the presentinvention are as follows:

[0072] (4Z,7Z,10Z,13Z,16Z,19Z)-N-(4-methylpiperazin-1-yl)docosahexaenoamide of the formula IV, optical isomers thereof, or theirpharmaceutically acceptable salts:

[0073] and(4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]docosahexaenoamideof the formula V, optical isomers thereof (for example, 2S-form,2R-form) or their pharmaceutically acceptable salts:

[0074] The stereoisomers in the present invention refer to thoseincluding any optical isomerism among (R)-forms, (S)-forms and racemicmodifications, and geometric isomerism among cis-form, trans-form, and amixture of them. As geometric isomerism, cis-form is preferred.

[0075] The pharmaceutically acceptable salts in the present inventioninclude salts with mineral acids such as sulfuric acid, hydrochloricacid and phosphoric acid, and salts with organic acids such as aceticacid, oxalic acid, lactic acid, tartaric acid, fumaric acid, maleicacid, methanesulfonic acid, and benzenesulfonic acid. Of these salts,the salts such as hydrochloride, citrate and maleate are preferred.

[0076] The compounds of the present invention can selectively suppressplatelet aggregation, especially, platelet aggregation caused bycollagen. As shown by the results of experiments to be described later,the compounds of the present invention have an ex vivo plateletaggregation suppressing effect which is more potent than that ofconventional GPIIb/IIIa receptor antagonists.

[0077] The compounds of the present invention can also suppressinflammation caused by inflammatory cytokines such as TNFα and PDGF.

[0078] The compounds of the present invention can be used in preventingor treating circulatory diseases.

[0079] The circulatory diseases herein refer to diseases in which thecirculatory state of the blood and lymph is impaired to cause disorderto tissue or cells. These diseases include all of those which occur fromvarious causes such as platelet aggregation and inflammatory cytokinessuch as TNFα and PDGF. Their examples include thrombotic diseases,arteriosclerotic diseases and hyperlipemic diseases.

[0080] The thrombotic diseases herein refer to states where the bloodvessel is obstructed by thrombus, and they are classified into arterialthrombosis and venous thrombosis. Arterial thrombus occurs mainly as acomplication of arteriosclerosis. Thrombus of the coronary arterybecomes the cause of myocardial infarction, and thrombus of the cerebralartery becomes the cause of cerebral infarction. Venous thrombosisincludes thrombosis of the superficial vein or deep vein, and deepvenous thrombosis, for example, is listed.

[0081] The thrombotic diseases include, for example, unstable angina,myocardial infarction, infarction associated with a prosthetic valve,obstruction of a grafted blood vessel after a coronary artery bypassoperation, transient cerebral ischemic attack, cerebral infarction,arteriosclerotic peripheral artery obstruction, erythromelalgia(thrombocythemia), thrombus of a hemodialysis shunt, angina of effort,restenosis after PTCA, and obstruction after blood vessel reconstructiveoperation (“Treatment of Thrombosis”, published by Medical Review, 1stEd. Jun. 20, 1996, author: Yasuo Ikeda).

[0082] The arteriosclerotic diseases herein refer to states where thearterial wall thickens and loses elasticity. Arteriosclerosis comes inthree types, atherosclerosis, Mönckeberg's arteriosclerosis, andarteriolosclerosis. Examples of the arteriosclerotic diseases includecerebral infarction and cerebral hemorrhage for the cerebral artery,ischemic heart diseases such as myocardial infarction and anginapectoris for the coronary artery, aortic aneurysm and aortic dissectionfor the aorta, nephrosclerosis and associated renal failure for therenal artery, and obstructive arteriosclerosis for the peripheralartery.

[0083] The hyperlipemic diseases herein refer to pathologic states whereserum cholesterol and/or triglyceride levels are increased. Examples arehypercholesterolemia and hyperlipidemia.

[0084] Each of the compounds in the present invention can beadministered orally or parenterally (as injection, external preparation,suppository, etc.). Its dose is preferably about 0.000001 to about 100mg/kg body weight/day, which is given as a single dose or severaldivided doses. More preferably, about 0.0001 to about 10 mg/kg bodyweight/day is given as a single dose or several doses per day. This dosemay be increased or decreased depending on the type of the disease orthe patient's age, body weight and symptoms.

[0085] To use the compounds of the present invention as pharmaceuticals,any forms, including a solid composition, a liquid composition and othercomposition, are available, and the optimal form is selected accordingto needs. Pharmaceutical compositions can be prepared by addingcustomary vehicles, bulking agents, binders, disintegration promoters,pH adjusting agents, and solubilizers to the compounds of the presentinvention, and forming the blends into tablets, pills, capsules,granules, powders, liquids, emulsions, suspensions, and injections bycustomary pharmaceutical techniques. Examples of the vehicles andbulking agents are lactose, magnesium stearate, starch, talc, gelatin,agar, pectin, gum arabic, olive oil, sesame oil, cacao butter, ethyleneglycol, and other ones in customary use.

[0086] To prevent the oxidation of the preparations, it is permissibleto add an antioxidant (tocopherol or the like), perform inclusion withan inclusion complexing agent such as cyclodextrin, or carry outencapsulation with a film of gelatin or the like.

[0087] Furthermore, the above compounds can be prepared, as described inUnexamined Japanese Patent Publication No. 1994-298642, in the form ofO/W type emulsions with the use of phospholipids or nonionic surfactantsas emulsifying agents. The emulsifying agents can be used alone or incombination of two or more, and the amount of their addition may be0.001 to 10% (W/V), preferably 0.01 to 5% (W/V), as desired.

[0088] As the phospholipids, soybean-derived phospholipid, eggyolk-derived phospholipid, lysolecithin, phosphatidylcholine (lecithin),and phosphatidylserine can be used alone or in combination. As thenonionic surfactants, the following can be preferably used alone or incombination, but without limitation: polyoxyethylene-polyoxypropyleneblock copolymers having a molecular weight of 500 to 15,000 (forexample, Pluronic F-68), polyalkylene glycols having a molecular weightof 1,000 to 10,000, polyoxyalkylene copolymers having a molecular weightof 1,000 to 20,000, hydrogenated castor oil polyoxyalkylene derivatives,castor oil polyoxyalkylene derivatives, glycerin esters of fatty acids,polyglycerin esters of fatty acids, sorbitan esters of fatty acids,polyoxyethylene castor oil, hydrogenated castor oil, polyoxyethylenealkyl ethers, and sucrose fatty acid esters.

[0089] The compounds of the present invention can be produced in thefollowing manner:

[0090] These compounds can be produced by an amidation method using, asthe starting materials, amines of the formula VI

[0091] wherein

[0092] l represents an integer of 0 to 10,

[0093] s represents 0 or 1, provided that when s is 0, p+q=4 or 5, butwhen s is 1, p+q=3 or 4, and in either case, either p or q is an integerof 1 or more,

[0094] R represents an alkyl group having 1 to 10 carbon atoms which maybe straight-chain or branched-chain, and

[0095] R^(A) represents hydrogen or an alkyl group having 1 to 10 carbonatoms which may be straight-chain or branched-chain,

[0096] and compounds of the formula VIII: A-CO₂—R′ wherein R′ representshydrogen or an alkyl group having 1 to 4 carbon atoms, and A representsan optionally substituted CH₃C_(n)CH_((2n−2m))— wherein n denotes aninteger of 4 to 22, and m represents an unsaturation number which is aninteger of 0 to 7.

[0097] The amines of the formula VI, as the starting material, can besynthesized in the usual manner.

[0098] The carboxylic acids or carboxylic acid esters of the formulaVIII, as the starting material, can be synthesized in the usual manner.The esters can be produced by an ordinary ester-forming reaction fromthe corresponding carboxylic acids or salts thereof. The correspondingcarboxylic acids or salts thereof may be synthetic substances ornaturally occurring substances. The synthetic substances areadvantageous in terms of economy, but the naturally occurring substancesare preferred because of lower toxicity. As the naturally occurringsubstances, those separated and purified from fish, etc., for example,can be included.

[0099] The carboxylic acids or carboxylic acid esters of the formulaVIII, which have substituents, may be naturally occurring products orsynthetic products.

[0100] The method of introducing the substituent during synthesis may bea method usually employed by one of ordinary skill in the art, forexample, the method of introducing the substituent into the carboxylicacid or ester of the formula VIII by a substitution or additionreaction.

[0101] If the substituent is an alkyl group, the alkyl group may beintroduced into CH₃C_(n)H_((2n−2m))COOH by use of an alkylating agent.

[0102] If the substituent is an OH group, the compounds at issue can besynthesized, without limitation, by hydroxylating naturally occurringDHA, and fractionating the hydroxylated DHA by HPLC or the like. Forexample, the compounds can be obtained by adding 10 to 200 mM of DHA, asa substrate, to a suspension of rainbow trout branchial cells orepithelial cells, mammalian platelets, or a human leukocyte-derivedestablished cell line such as RBL-1, and reacting the mixture at 10 to37° C. for 1 to 50 minutes. The reaction solution is acidified (withformic acid, acetic acid or trichloroacetic acid) to terminate thereaction. Then, the respective OH derivatives can be extracted with anorganic solvent (chloroform, methanol, ethyl acetate, acetonitrile,etc.), and fractionated by a method, such as HPLC or thin layerchromatography, using a development solvent (chloroform, methanol, ethylacetate, acetonitrile, water, or trifluoroacetic acid). However, thesemethods are not limitative. The respective OH derivatives can also beprepared by selective methods of synthesis using site-specific enzymes.The derivatives, such as 4-OH-DHA, 10-OH-DHA, 11-OH-DHA, 14-OH-DHA,8-OH-DHA, and 17-OH-DHA (S-forms thereof) are commercially availablefrom Wako Pure Chemical Industries.

[0103] In obtaining the starting materials by synthesis, the amines ofthe formula VI or the carboxylic acids or esters of the formula VIII maybe separated, or can be used dissolved in solvents.

[0104] The amidation method is not limited, but the targeted compoundcan generally be synthesized by the mixed acid anhydride method. Forexample, the following methods can be named here:

[0105] (1) Weinreb Method

[0106] The compound of the present invention can be produced by reactingthe ester of the formula VIII with the reaction product formed betweenthe amine of the formula VI and a trialkylaluminum, especially (CH₃)₃Al.This reaction will be described in detail using the following scheme(for convenience's sake, the scheme shows the ester of the formula VIIIas not substituted by a substituent):

[0107] The reaction for formation of the compound VII in the above firststep is performed by reacting the amine of the formula VI (preferably,an acid-addition salt such as a hydrochloride) with (CH₃)₃Al. Thisreaction is preferably carried out, with cooling, in an aromatichydrocarbon solvent (for example, toluene, xylene or benzene).

[0108] At this time, the amount of (CH₃)₃Al is preferably 0.5 to 5.0equivalents per equivalent of the compound VI.

[0109] The above second step is performed by reacting the compound VII,obtained in the first step, with the ester of the formula VIII. Thisreaction is preferably carried out, with heating, in an aromatichydrocarbon solvent (for example, toluene, xylene or benzene). Thereaction temperature is preferably 40 to 70° C. The reaction temperaturepreferably does not exceed about 70° C., because the exceedingtemperature would make the product decomposable. The reaction time ispreferably 1 to 5 hours.

[0110] At this time, the amount of the ester of the formula VIII ispreferably 0.5 to 5.0 equivalents per equivalent of the compound VII.

[0111] (2) Method Using (COCl)₂

[0112] The compound of the present invention can also be obtained byreacting the amine of the formula VI

[0113] wherein p, q, s, l, R and R^(A) are as defined above, with anacid chloride formed by the reaction between the carboxylic acid of theformula VIII: A-CO—OH [wherein A represents an optionally substitutedCH₃C_(n)H_((2n−2m))— (wherein n denotes an integer of 4 to 22, and mrepresents an unsaturation number which is an integer of 0 to 7)] and(COCl)₂.

[0114] The above first step—the reaction for formation of the acidchloride by the reaction between the carboxylic acid of the formula VIIIand (COCl)₂—is preferably carried out, with cooling, in an aromatichydrocarbon solvent (for example, toluene, xylene or benzene).

[0115] At this time, the amount of (COCl)₂ is preferably 1 to 5equivalents per equivalent of the carboxylic acid of the formula VIII:A-CO—OH.

[0116] The above second step—the reaction between the acid chlorideobtained in the first step and the amine of the formula VI—is preferablyperformed in a hydrocarbon solvent (for example, dichloromethane orchloroform) or an aromatic hydrocarbon solvent (for example, toluene,xylene or benzene). The reaction temperature is preferably −5 to +5° C.The reaction temperature preferably does not exceed about +5° C.,because the exceeding temperature would make the product decomposable.The reaction time is preferably 0.5 to 5 hours.

[0117] At this time, the amount of the amine of the formula VI ispreferably 1 to 5 equivalents per equivalent of the acid chloride.

[0118] Whichever method of production is employed, the compound of thepresent invention can be isolated and purified in the usual manner (e.g.filtration, solvent extraction, recrystallization, reprecipitation orchromatography), if desired, after completion of the reaction.

[0119] The stereoisomer can be obtained by selecting suitable startingmaterials. In the case of a mixture of stereoisomers, stereochemicallypure isomers can be obtained by chromatography or racemic resolution.

EXAMPLES

[0120] The present invention will now be described in further detail byExamples and Test Examples, which in no way limit the technical scope ofthe present invention.

Example 1 Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-N-(4-methylpiperazin-1-yl)docosahexaenoamide(Compound 1)

[0121] Toluene (15 ml), dried using the molecular sieve MS 4A, was mixedwith 10.3 ml of a n-hexane solution of 15% Me₃Al. With the mixture beingcooled in an ice-methanol bath, 1.69 ml (14.1 mmols) of1-amino-4-methylpiperazine was added dropwise over about 2 minutes(inner temperature <7° C.). The 1-amino-4-methylpiperazine was finallywashed using 2 ml of toluene. After stirring for 35 minutes, thetemperature was raised to room temperature, and 6 ml of a toluenesolution of 5.0 g (14.0 mmols) of docosahexaenoic acid ethyl ester(hereinafter referred to as DHA ethyl ester) was added dropwise over 6minutes (inner temperature 25˜26° C.). After stirring for 2 hours at 70°C., the mixture was cooled with ice, and 24 ml of 0.67N hydrochloricacid was added dropwise (inner temperature raised up to 39° C.). Themixture was stirred for 10 minutes, and water and ethyl acetate wereadded. Then, the mixture was filtered through Celite, the filtrate wasseparated into respective layers, and the organic layer was washed twicewith 20 ml of a saturated aqueous solution of sodium chloride. Afterthis layer was dried over anhydrous sodium sulfate, it was concentratedunder reduced pressure on a 32° C. water bath to obtain the captionedcompound. This compound was subjected to silica gel columnchromatography (mobile phase: CHCl₃→AcOEt→CHCl₃:MeOH (4:1)), and thenfurther subjected to silica gel column chromatography (mobile phase:CHCl₃:MeOH (19:1)) for purification. NMR confirmed the purified compoundto have the following structure (yield 5.4 g, purity 95%):

δ (ppm) J (Hz) Proton No. 5.45-5.26 m 12H 4, 5, 7, 8, 10, 11, 13, 14,16, 17, 19, 20 2.90-2.75 m 14H 6, 9, 12, 15, 18, 21, 2′, 3′, 5′, 6′2.70-2.50 m 4H 2′, 3′, 5′, 6′ 2.42-2.36 m 2H 3 2.33 s 3H N—Me 2.16 t 7.62H 2 2.12-2.03 m 2H 21 0.98 t 7.6 3H 22

Example 2 Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]docosahexaenoamide(Compound 2)

[0122] Toluene (15 ml), dried using MS 4A, was mixed with 10.3 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 2.03 ml (14.1 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 2minutes (inner temperature −7→+8° C.). The2-(2-aminoethyl)-1-methylpyrrolidine was finally washed using 2 ml oftoluene. After stirring for 20 minutes, the temperature was raised toroom temperature, and 6 ml of a toluene solution of 5.0 g (14.0 mmols)of DHA ethyl ester was added dropwise over 2 minutes (inner temperature28˜29° C.). After stirring for 2 hours at 70° C., the mixture was cooledwith ice, and 24 ml of 0.67N hydrochloric acid was added dropwise (innertemperature 12˜27° C.). The mixture was stirred for 10 minutes, andwater and ethyl acetate were added. Then, the mixture was filteredthrough Celite, the filtrate was separated into respective layers withthe addition of a small amount of NaOH, and the organic layer was washedtwice with 20 ml of a saturated aqueous solution of sodium chloride.After this layer was dried over anhydrous sodium sulfate, it wasconcentrated under reduced pressure on a 32° C. water bath to obtain thecaptioned compound (yield 4.7 g). This compound was subjected to silicagel column chromatography (mobile phase: CHCl₃:MeOH (19:1˜9:1)) forpurification. NMR confirmed the purified compound to have the followingstructure:

δ (ppm) J (Hz) Proton No. 6.75 brs 1H NH 5.45-5.25 m 12H 4, 5, 7, 8, 10,11, 13, 14, 16, 17, 19, 20 3.46 sextet 6.8 1H 1′ 3.27-3.18 m 1H 1′ 3.05ddd 2.6, 6.8, 8.8 1H 2″ 2.90-2.75 m 10H 6, 9, 12, 15, 18, 21 2.40 q 7.02H 3 2.31 s 3H 1″(═N—Me) 2.28-2.03 m 6H 2, 21, 5″ 1.96-1.86 m 1H 3″(or4″) 1.82-1.65 m 3H 3″ and/or 4″ and/or 2′ 1.64-1.52 m 2H 3″ and/or 4″and/or 2′ 0.98 t 7.6 3H 22

Example 3 Synthesis of N-[2-(1-methylpyrrolidin-2-yl)ethyl]caprylamide(Compound 3)

[0123] Toluene (1.0 ml), dried using MS 4A, was mixed with 1.48 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 0.25 ml (1.16 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 2minutes. After stirring for 20 minutes, the temperature was raised toroom temperature, and 0.5 ml of a toluene solution of 0.2 g (1.16 mmols)of caprylic acid ethyl ester was added dropwise over 1 minute. Afterstirring for 2 hours at 70° C., the mixture was cooled with ice, and 10ml of 1N NaOH was added dropwise. The mixture was stirred for 10minutes, and water and ethyl acetate were added. Then, the mixture wasseparated into respective layers, and the organic layer was washed twicewith 20 ml of a saturated aqueous solution of sodium chloride. Afterthis layer was dried over anhydrous magnesium sulfate, it wasconcentrated under reduced pressure on a 30° C. water bath to obtain thecaptioned compound (yield 0.22 g). This compound was subjected to silicagel column chromatography (mobile phase: CHCl₃:MeOH (19:1→9:1)) forpurification. NMR and mass spectrum confirmed the purified compound tohave the following structure:

Molecular weight: Chemical 254.41 shift Proton No. Assigned to: m/zAssigned to: 0.88 3(t, J=6.8 Hz) H-8 254 (M⁺) — 1.29 8(m) —CH ₂— 239(M⁺-15) CH₃ 1.59 2(m)

170 (M⁺-84) C₅H₁₀N 2.14 2(m) H-2 155 (M⁺-99) C₇H₁₅ 2.31 3(sz)

84 (M⁺-170) C₁₀H₂₀NO 3.46 1(q, J=6.7 Hz)

6.71 1(s) —NH—

Example 4 Synthesis of N-[2-(1-methylpyrrolidin-2-yl)ethyl]myristamide(Compound 4)

[0124] Toluene (1.00 ml), dried using MS 4A, was mixed with 1.00 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 0.17 ml (0.78 mmol) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 2minutes. After stirring for 20 minutes, the temperature was raised toroom temperature, and 0.5 ml of a toluene solution of 0.2 g (0.78 mmol)of myristic acid ethyl ester was added dropwise over 1 minute. Afterstirring for 2 hours at 70° C., the mixture was cooled with ice, and 1NNaOH solution was added dropwise. The mixture was stirred for 10minutes, and water and ethyl acetate were added. Then, the mixture wasseparated into respective layers, and the organic layer was washed twicewith 20 ml of a saturated aqueous solution of sodium chloride. Afterthis layer was dried over anhydrous magnesium sulfate, it wasconcentrated under reduced pressure on a 30° C. water bath to obtain thecaptioned compound (yield 0.25 g). This compound was subjected to silicagel column chromatography (mobile phase: CHCl₃:MeOH (19:1→9:1)) forpurification. NMR and mass spectrum confirmed the purified compound tohave the following structure:

Chemical Molecular weight: 338.566 shift Proton No. Assigned to: m/zAssigned to: 0.88 3(t, J=6.6 Hz) H-14 388 (M⁺) — 1.27 20(m) —CH ₂— 323(M⁺-15) CH₃ 1.59 2(m)

170(M⁺-168) C₉H₁₆N₂O 2.14 2(m) H-2 84 (M⁺-254) C₁₆H₃₃NO 2.31 3(s)

3.46 1(q, J=6.6 Hz)

Example 5 Synthesis of 9Z-N-[2-(1-methylpyrrolidin-2-yl)ethyl]oleamide(Compound 5)

[0125] Toluene (18 ml), dried using MS 4A, was mixed with 12.3 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 2.42 ml (16.7 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 5minutes. The 2-(2-aminoethyl)-1-methylpyrrolidine was finally washedusing 2 ml of toluene. After stirring for 20 minutes, the temperaturewas raised to room temperature, and 7 ml of a toluene solution of 5.0 g(16.9 mmols) of oleic acid methyl ester was added dropwise over 2minutes. After stirring for 2.5 hours at 70° C., the mixture was cooledwith ice, and 30 ml of 0.67N hydrochloric acid was added dropwise. Anaqueous solution of 1N NaOH (about 100 ml) was added, and the mixturewas extracted with about 100 ml of ethyl acetate. At this time, the pHof the aqueous layer was 9 to 10. The organic layer was washed twicewith 20 ml of a saturated aqueous solution of sodium chloride. Afterthis layer was dried over anhydrous sodium sulfate, it was concentratedunder reduced pressure on a 32° C. water bath to obtain the captionedcompound. This compound was subjected to silica gel columnchromatography (BW80S 150 g, FUJISILYSIA, mobile phase: CHCl₃:MeOH(9:1→4:1→3:1 (V/V))) for purification. The purified compound wasconcentrated under reduced pressure on a 35° C. water bath to obtain4.09 g of a pale yellow liquid (10.4 mmols, yield 62%). NMR confirmedthis product to have the following structure:

δ (ppm) J (Hz) Proton No. 6.78 brs 1H NH 5.40-5.28 m 2H 9, 10 3.46sextet 6.8 1H 1′ 3.28-3.18 m 1H 1′ 3.07 ddd 2.8, 6.8, 8.8 1H 2″ 2.34 s3H 1″ (═Me) 2.33-2.21 m 1H 5″ 2.21-2.10 m 3H 2, 5″ 2.03-1.97 m 4H 8, 111.98-1.85 m 1H 3″ (or 4″) 1.80-1.70 m 3H 3″, 4″ 1.68-1.56 m 4H 3, 2′1.38-1.20 m 20H 4-7, 12-17 0.89 t 7.6 3H 18

Example 6 Synthesis of(9Z,12Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]linoleamide (Compound 6

[0126] Toluene (0.7 ml), dried using MS 4A, was mixed with 0.83 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 0.14 ml (0.98 mmol) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 2minutes. After stirring for 20 minutes, the temperature was raised toroom temperature, and 0.4 ml of a toluene solution of 0.2 g (0.65 mmol)of linoleic acid ethyl ester was added dropwise over 1 minute. Afterstirring for 2 hours at 70° C., the mixture was cooled with ice, and 10ml of a 1N NaOH solution was added dropwise (inner temperature 12˜27°C.). The mixture was stirred for 10 minutes, and water and ethyl acetatewere added. Then, the mixture was separated into respective layers, andthe organic layer was washed twice with 20 ml of a saturated aqueoussolution of sodium chloride. After this layer was dried over anhydrousmagnesium sulfate, it was concentrated under reduced pressure on a 30°C. water bath to obtain the captioned compound (yield 0.114 g). Thiscompound was subjected to silica gel column chromatography (mobilephase: CHCl₃:MeOH (19:1→9:1)) for purification. NMR and mass spectrumconfirmed the purified compound to have the following structure:

Chemical Molecular weight: 390.638 shift Proton No. Assigned to: m/zAssigned to: 0.89 3(t, J=6.8 Hz) H-18 390 (M⁺) — 1.31 14(m) —CH ₂—84(M⁺-304) C₂₀H₃₆NO 1.58 2(m)

2.06 2(qn, J=7.3 Hz) H-17 2.14 2(t, J=3.4 Hz) H-2 2.31 3(s)

2.77 2(m) ═CHCH ₂CH═ 3.46 1(q, J=6.6 Hz)

5.36 4(m) —CH═CH— 6.70 1(s) —NH—

Example 7 Synthesis of(9Z,12Z,15Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]linolenamide (Compound7)

[0127] Toluene (0.6 ml), dried using MS 4A, was mixed with 1.99 ml of ann-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 0.23 ml (1.56 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 1minute. After stirring for 20 minutes, the temperature was raised toroom temperature, and 0.2 ml of a toluene solution of 0.2 g (0.65 mmol)of linolenic acid ethyl ester was added dropwise over 1 minute. Afterstirring for 2 hours at 70° C., the mixture was cooled with ice, and 10ml of a 1N NaOH solution was added dropwise. The mixture was stirred for10 minutes, and water and ethyl acetate were added. Then, the mixturewas separated into respective layers, and the organic layer was washedtwice with 20 ml of a saturated aqueous solution of sodium chloride.After this layer was dried over anhydrous magnesium sulfate, it wasconcentrated under reduced pressure on a 30° C. water bath to obtain thecaptioned compound (yield 0.13 g). This compound was subjected to silicagel column chromatography (mobile phase: CHCl₃:MeOH (19:1→9:1)) forpurification. NMR and mass spectrum confirmed the purified compound tohave the following structure:

Chemical Molecular weight: 338.622 shift Proton No. Assigned to: m/zAssigned to: 0.98 3(t, J=7.5 Hz) H-18 388(M⁺) — 1.26 8(m) —CH ₂— 84(M⁺-304) C₂₀H₃₄NO 1.59 2(m)

2.06 2(m) H-17 2.14 2(m) H-2 2.32 3(s)

2.81 4(m) ═CHCH ₂CH═ 3.46 1(q, J=6.6 Hz)

5.37 6(m) —CH═CH— 6.79 1(s) —NH—

Example 8 Synthesis of(5Z,8Z,11Z,14Z,17Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]eicosapentaenoamide(Compound 8)

[0128] Toluene (18 ml), dried using MS 4A, was mixed with 12.3 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 2.42 ml (16.7 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine was added dropwise over about 5minutes. After stirring for 20 minutes, the temperature was raised toroom temperature, and 7 ml of a toluene solution of 4.5 g (13.6 mmols)of eicosapentaenoic acid ethyl ester was added dropwise over 2 minutes.After stirring for 2.5 hours at 70° C., the mixture was cooled with ice,and 30 ml of 0.67N hydrochloric acid was added dropwise. An aqueoussolution of 1N NaOH (about 100 ml) was added, and the mixture wasextracted with about 100 ml of ethyl acetate. At this time, the pH ofthe aqueous layer was 9 to 10. The organic layer was washed twice with20 ml of a saturated aqueous solution of sodium chloride. After thislayer was dried over anhydrous sodium sulfate, it was concentrated underreduced pressure on a 32° C. water bath to obtain the captionedcompound. This compound was subjected to silica gel columnchromatography (BW-80S 150 g, FUJISILYSIA, mobile phase: CHCl₃:MeOH(9:1→4:1→3:1 (V/V))) for purification. The purified compound wasconcentrated under reduced pressure on a 35° C. water bath to obtain 3.2g (yield 57%) of a pale yellow liquid. NMR confirmed this product tohave the following structure:

δ (ppm) J (Hz) Proton No. 6.70 brs 1H NH 5.45-5.28 m 10H 5, 6, 8, 9, 11,12, 14, 15, 17, 18 3.47 sextet 6.8 1H 1′ 3.28-3.18 m 1H 1′ 3.08 ddd 2.8,6.8, 8.8 1H 2″ 2.90-2.88 m 8H 7, 10, 13, 16 2.33 s 3H 1″ (═Me) 2.33-2.20m 1H 5″ 2.20-2.03 m 7H 2, 4, 19, 5″ 1.98-1.85 m 1H 3″ (or 4″) 1.80-1.65m 7H 3, 2′, 3″, 4″ 0.98 t 7.6 3H 20

Example 9 Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-N-[(1-methylpyrrolidin-2-yl)methyl]docosahexaenoamide(Compound 9)

[0129] Toluene (5 ml), dried using MS 4A, was mixed with 3.2 ml of ann-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, a toluene solution (2 ml) of 0.6 g of2-aminomethyl-1-methylpyrrolidine was added dropwise over about 2minutes (inner temperature −10→+0° C.). After stirring for 20 minutes,the temperature was raised to 12° C., and 2 ml of a toluene solution of1.56 g (4.38 mmols) of DHA ethyl ester was added dropwise over 2 minutes(inner temperature 10˜13° C.). After stirring for 2.5 hours at 70° C.,the mixture was cooled with ice, and 7.5 ml of 0.67N hydrochloric acidwas added dropwise. After 35 ml of 1N NaOH was added, the mixture wasextracted twice with ethyl acetate, followed by washing the extracttwice with 20 ml of water and 20 ml of a saturated aqueous solution ofsodium chloride. The washed product was dried over anhydrous sodiumsulfate, and then concentrated under reduced pressure on a 40° C. waterbath to obtain the captioned compound. This compound was subjected tosilica gel column chromatography (mobile phase: CHCl₃:MeOH (50:1→9:1))for purification (1.4 g, 3.3 mmols, yield 75%). NMR confirmed thepurified compound to have the following structure:

δ (ppm) J (Hz) Proton No. 6.0 brs 1H NH 5.45-5.25 m 12H 4, 5, 7, 8, 10,11, 13, 14, 16, 17, 19, 20 3.61 ddd 2.4, 7.6, 1H 1′ 13.6 3.20-3.00 m 2H1′, 2″ 2.90-2.75 m 10H 6, 9, 12, 15, 18, 21 2.45-2.33 M 3H 3, 5″ 2.30 s3H 1″(═N—Me) 2.28-2.17 m 3H 2, 5″ 2.12-2.03 m 2H 21 1.84 dq 11.2, 7.6 1H3″ (or 4″) 1.75-1.62 m 2H 4″ (or 3″) 1.60-1.51 m 1H 3″ (or 4″) 0.98 t7.6 3H 22

Example 10 Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]docosahexaenoamide(Compound 10)

[0130] Crude docosahexaenoic acid (DHA) (2.1 g) and 10 ml of toluenewere mixed. With the mixture being cooled with ice, 0.93 ml (10.6 mmols)of (COCl)₂ was added, and the mixture was stirred for 2 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure on a 40° C. water bath and, after addition of toluene, wasconcentrated again under reduced pressure to obtain a crude DHA acidchloride as a yellow liquid. After combining 3 ml (21 mmols) of2-(2-aminoethyl)-1-methylpyrrolidine with 20 ml of CH₂Cl₂, the total ofthe crude DHA acid chloride was added under cooling with ice. Themixture was stirred for 1 hour while being cooled with ice, and 30 ml ofCH₂Cl₂ and 50 ml of iced water were added. The organic layer wasseparated using a separating funnel, washed with 130 ml of 1M HCl, andwashed twice with water. The washed layer was dried over Na₂SO₄, andsubjected to silica gel column chromatography (50 g silica, mobilephase: n-hexane:ethyl acetate (9:1→3:1)) for purification, therebyobtaining 1.9 g (yield 73%) of a light yellow liquid. NMR confirmed thepurified product to be the same compound as in Example 2.

Example 11 Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-N-[3-(1-methylpyrrolidin-2-yl)propyl]docosahexaenoamide(Compound 11)

[0131] Toluene (1.34 ml), dried using MS 4A, was mixed with 0.86 ml of an-hexane solution of 15% Me₃Al. With the mixture being cooled in anice-methanol bath, 0.167 g of 2-(3-aminopropyl)-1-methylpyrrolidine in0.6 ml of toluene was added dropwise over about 1 minute (innertemperature −15° C.). After stirring for 20 minutes, the temperature wasraised to 20° C., and 0.6 ml of a toluene solution of 0.42 g (1.2 mmols)of DHA ethyl ester was added dropwise (inner temperature 20° C.). Afterstirring for 3 hours at 70° C., the mixture was cooled with ice, and 2.1ml of 0.67N hydrochloric acid was added dropwise. After 10 ml of anaqueous solution of 1N NaOH was added, the mixture was extracted withethyl acetate, followed by washing the extract with water and asaturated aqueous solution of sodium chloride. The washed product wasdried over anhydrous sodium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatography(mobile phase: CHCl₃:MeOH (50:1→9:1→4:1)), thereby obtaining 0.21 g ofthe captioned compound (amount yielded: 0.46 mmol, yield: 39%, purity:97.9%). NMR confirmed the purified compound to have the followingstructure:

δ (ppm) J (Hz) Proton No. 6.0 brs 1H NH 5.45-5.25 m 12H 4, 5, 7, 8, 10,11, 13, 14, 16, 17, 19, 20 3.37-3.28 m 1H 1′ 3.23-3.13 m 1H 1′ 3.08 ddd2.4, 7.6, 9.2 1H 2″ 2.90-2.75 m 10H 6, 9, 12, 15, 18, 21 2.41 q 7.6 2H 32.31 s 3H 1″ (═N—Me) 2.21 t 7.6 2H 2 2.20-2.0 m 2H 5″ 2.12-2.03 m 2H 211.96-1.86 m 1H 3″ (or 4″) 1.82-1.63 m 3H 4″ (or 3″), 2′ (or 3′)1.60-1.41 m 3H 3″ (or 4″), 2′ (or 3′) 1.35-1.24 m 1H 2′ (or 3′) 0.98 t7.6 3H 22

Example 12 Synthesis ofN-[2-((2S)-1-methylpyrrolidin-2-yl)ethyl](4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoamide(2S-form of Compound 2)

[0132] (1) (5S)-5-[(tert-butyldimethysilyloxy)methyl]pyrrolidin-2-one

[0133] Imidazole (4.09 g, 60 mmols) and 4-dimethylaminopyridine (100 mg,0.82 mmol) were added to a CH₂Cl₂ (85 ml) solution of(5S)-5-(hydroxymethyl)-2-pyrrolidinone (3.14 g, 27.3 mmols). Then, withthe mixture being cooled with ice, a CH₂Cl₂ (15 ml) solution oftert-butyldimethylsilyl chloride (4.53 g, 30 mmols) was added, and themixture was stirred for 15 hours at room temperature. The solvent wasdistilled off under reduced pressure, and then the residue was subjectedto silica gel column chromatography (SiO₂ 300 g, MeOH—CHCl₃ 5:95 v/v) toobtain (5S)-5-[(tert-butyldimethylsilyloxy)methyl]pyrrolidin-2-one (6.22g, 99.5%): [α]_(D) ²⁷+42.32 (c 1.136, CHCl₃). IR ν max (film) cm⁻¹:3242, 1697. ¹H-NMR (CDCl₃)δ: 5.79 (1H, br), 3.80-3.71 (1H, m), 3.63 (1H,dd, J=9.9, 3.8 Hz), 3.44 (1H, dd, J=9.9, 7.7 Hz), 2.44-2.26 (2H, m),2.24-2.11 (1H, m), 1.80-1.67 (1H, m), 0.89 (9H, s), 0.06 (6H, s). MSm/z: 230 (M⁺+1), 214 (M⁺−15), 172 (100%). HRMS: Calculated forC₁₀H₂₀NO₂Si: 214.1263 (M⁺−15). Found: 214.1240 (M⁺−15).

[0134] (2)(5S)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one

[0135] A THF (10 ml) solution of the(5S)-5-[(tert-butyldimethylsilyloxy)methyl]pyrrolidin-2-one (3.71 g,16.2 mmols) was added dropwise to a THF (70 ml) suspension of NaH (60%oil disp. 778 mg, 19.4 mmols), with the system being cooled with ice,whereafter the mixture was stirred for 30 minutes at room temperature.To the mixture, MeI (5.0 ml, 81 mmols) was added during cooling withice, and the mixture was stirred for 15 hours at room temperature.During cooling with ice, a saturated aqueous solution (50 ml) of NH₄Clwas added, and the solvent was distilled off under reduced pressure. Theresidue was diluted with water, whereafter the dilution was extractedwith AcOEt (50 ml×3), and the organic layer was dried over MgSO₄. Thesolvent was distilled off under reduced pressure, and then the residuewas subjected to silica gel column chromatography (SiO₂ 150 g,MeOH—CHCl₃ 3:97 v/v) to obtain(5S)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one(3.86 g, 97.9%): [α]_(D) ²⁹+3.46 (c 1.13, CHCl₃). IR ν max (film) cm⁻¹:1682. ¹H-NMR (CDCl₃)δ: 3.73 (1H, dd, J=10.2, 3.3 Hz), 3.60 (1H, dd,J=10.2, 4.1 Hz), 3.55 (1H, quint, J=4.1 Hz), 2.85 (3H, s), 2.50-2.37(1H, m), 2.35−2.23 (1H, m), 2.15-2.01 (1H, m), 1.89-1.77 (1H, m), 0.89(9H, s), 0.06 (3H, s), 0.05 (3H, s). MS m/z: 228 (M⁺−15), 186, 98(100%). HRMS: Calculated for C₁₁H₂₂NO₂Si: 228.1420 (M⁺−15). Found:228.1441 (M⁺−15).

[0136] (3) (5S)-5-(bromomethyl)-1-methylpyrrolidin-2-one

[0137] Bu₄NF (1 mol THF solution, 17.5 ml, 17.5 mmols) was added to aTHF (100 ml) solution of the(5S)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one(3.84 g, 15.8 mmols), and the mixture was stirred for 15 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure, and then the residue was subjected to silica gel columnchromatography (SiO₂ 150 g, MeOH—CHCl₃ 1:9 v/v) to obtain(5S)-5-(hydroxymethyl)-1-methylpyrrolidin-2-one as a mixture (2.46 g)with inseparable impurities.

[0138] Ph₃P (4.13 g, 15.75 mmols) was added to a CH₃CN (35 ml) solutionof the above mixture (2.46 g), and then a CH₃CN (10 ml) solution of CBr₄(5.22 g, 15.75 mmols) was added dropwise during cooling with ice,followed by stirring the mixture for 15 hours at room temperature. Thereaction mixture was concentrated under reduced pressure, and then theresidue was subjected to silica gel column chromatography (SiO₂ 150 g,AcOEt) to obtain (5S)-5-(bromomethyl)-1-methylpyrrolidin-2-one (2.57 g,84.7%): IR ν max (film) cm⁻¹: 1688. ¹H-NMR (CDCl₃)δ: 3.81 (1H, sextet,J=4.1 Hz), 3.53 (1H, d, J=4.1 Hz), 2.84 (3H, s), 2.62-2.46 (1H, m),2.42-2.27 (1H, m), 2.26-2.14 (1H, m), 2.01-1.88 (1H, m). MS m/z: 193(M⁺+2), 191 (M⁺), 98 (100%). HRMS: Calculated for C₆H₁₀NOBr: 190.9945(M⁺). Found: 190.9935 (M⁺).

[0139] (4) 2-((2S)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile

[0140] KCN (886 mg, 13.6 mmols), NaCN (666 mg, 13.6 mmols) and18-crown-6 (207 mg, 1.02 mmols) were added to a CH₃CN (25 ml) solutionof the (5S)-5-(bromomethyl)-1-methylpyrrolidin-2-one (1.31 g, 6.8mmols), and the mixture was heated for 43 hours under reflux. Theinorganic matter was filtered off, and the filtrate was diluted withAcOEt (50 ml). The dilution was washed with a saturated solution (30 ml)of NaCl, and dried over MgSO₄. The solvent was distilled off underreduced pressure, and then the residue was subjected to silica gelcolumn chromatography (SiO₂ 50 g, MeOH—CHCl₃ 1:9 v/v) to obtain2-((2S)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile as a mixture (1.05g) with impurities difficult to separate: IR ν max (film) cm⁻¹: 2246,1685. ¹H-NMR (CDCl₃)δ: 3.85-3.76 (1H, m), 2.88 (3H, s), 2.68 (1H, dd,J=17.0, 4.4 Hz), 2.61 (1H, dd, J=17.0, 6.0 Hz), 2.62-2.48 (1H, m),2.46-2.30 (2H, m), 2.00-1.86 (1H, m). MS m/z: 138 (M⁺), 98 (100%). HRMS:Calcd. for C₇H₁₀N₂O: 138.0793 (M⁺). Found: 138.0794 (M⁺).

[0141] (5) 2-((2S)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile

[0142] Lawesson's reagent (Tokyo Kasei Kogyo) (1.54 g, 3.8 mmols) wasadded to a benzene (25 ml) solution of the2-((2S)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile (1.05 g, 7.6 mmols),and the mixture was heated for 2 hours under reflux. The solvent wasdistilled off under reduced pressure, and then the residue was subjectedto silica gel column chromatography (SiO₂ 60 g, MeOH—CHCl₃ 1:9 v/v) toobtain 2-((2S)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile (941 mg,80.6%): IR ν max (film) cm⁻¹: 2246. ¹H-NMR (CDCl₃)δ: 4.20-4.10 (1H, m),3.30 (3H, s), 3.25-2.98 (2H, m), 277 (1H, dd, J=17.0, 4.9 Hz), 2.68 (1H,dd, J=17.0, 6.6 Hz), 2.50-2.36 (1H, m), 2.06-1.94 (1H, m). MS m/z: 154(M⁺), 114 (100%). HRMS: Calcd. for C₇H₁₀N₂S: 154.0565 (M⁺). Found:154.0561 (M⁺).

[0143] (6)N-[2-((2S)-1-methylpyrrolidin-2-yl)ethyl](4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoamide

[0144] Raney-Ni (1.5 ml) was added to an EtOH (30 ml) solution of the2-((2S)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile (940 mg, 6.1mmols), and the mixture was heated for 24 hours under reflux (upon TCL,the starting materials had not completely disappeared). The inorganicmatter was filtered off, and then the solvent was distilled off underreduced pressure. The residue was subjected to silica gel columnchromatography (SiO₂ 40 g, MeOH—CHCl₃ 1:9 v/v) to obtain2-((2S)-1-methylpyrrolidin-2-yl)ethanenitrile as a mixture (195 mg) withimpurities difficult to separate.

[0145] To a THF (10 ml) suspension of LiAlH₄ (88 mg, 2.3 mmols), a THF(5 ml) solution of the 2-((2S)-1-methylpyrrolidin-2-yl)ethanenitrilemixture (195 mg) was added dropwise little by little, with stirring,while being cooled with ice. Then, the mixture was heated under refluxfor 15 minutes. After cooling, c. NH4OH was added, and the reactionmixture was filtered using Celite. The filtrate was distilled underreduced pressure to remove the solvent, whereafter CH₂Cl₂ was added tothe residue, and the mixture was dried over K₂CO₃. The solvent wasdistilled off under reduced pressure. CH₂Cl₂ (8 ml) was added to theresulting crude product, 2-((2S)-1-methylpyrrolidin-2-yl)ethylamine,then a CH₂Cl₂ (2 ml) solution of DHA-Cl (150 mg, 0.43 mmol) was added,and then the mixture was stirred for 15 hours at room temperature. Thereaction mixture was concentrated under reduced pressure, and then theresidue was subjected to silica gel column chromatography (SiO₂ 30 g,MeOH—CHCl₃ 1:9 v/v saturated with a concentrated aqueous solution ofNH₄OH) to obtain the captioned compound (157 mg, 22.4%): [α]_(D) ²⁴−6.85(c 0.928, CHCl₃). ¹H-NMR (CDCl₃)δ: 6.71 (1H, br), 5.45-5.26 (12H, m),3.52-3.40 (1H, m), 3.28-3.16 (1H, m), 3.09-3.01 (1H, m), 2.90-2.77 (10H,m), 22.44-2.35 (2H, m), 2.31 (3H, s), 2.29-2.02 (6H, m), 1.97-1.82 (1H,m), 1.80-1.51 (5H, m), 0.97 (3H, t, J=7.7 Hz).

Example 13 Synthesis ofN-[2-((2R)-1-methylpyrrolidin-2-yl)ethyl](4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoamide(2R-form of Compound 2)

[0146] (1) (5R)-5-[(tert-butyldimethylsilyloxy)methyl]pyrrolidin-2-one

[0147] Imidazole (5.24 g, 77 mmols) was added to a CH₂Cl₂ (85 ml)solution of (5R)-5-(hydroxymethyl)-2-pyrrolidinone (4.03 g, 35 mmols).Then, during cooling with ice, a CH₂Cl₂ (15 ml) solution oftert-butyldimethylsilyl chloride (4.53 g, 30 mmols) was added, and themixture was stirred for 15 hours at room temperature. The solvent wasdistilled off under reduced pressure, and then the residue was subjectedto silica gel column chromatography (SiO₂ 300 g, MeOH—CHCl₃ 5:95 v/v) toobtain (5R)-5-[(tert-butyldimethylsilyloxy)methyl]pyrrolidin-2-one (7.92g, 98.7%):

[0148] [α]_(D) ²⁵−47.44 (c 1.80, CHCl₃).

[0149] (2)(5R)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one

[0150] A THF (20 ml) solution of the(5R)-5-[(tert-butyldimethylsilyloxy)methyl]pyrrolidin-2-one (5.73 g, 25mmols) was added dropwise, during cooling with ice, to a THF (100 ml)suspension of NaH (60% oil disp. 1.20 g, 30 mmols), whereafter themixture was stirred for 30 minutes at room temperature. To the mixture,MeI (7.8 ml, 125 mmols) was added during cooling with ice, and themixture was stirred for 15 hours at room temperature. During coolingwith ice, a saturated aqueous solution (50 ml) of NH₄Cl was added, andthe solvent was distilled off under reduced pressure. The residue wasdiluted with water, whereafter the dilution was extracted with AcOEt (50ml×3), and the organic layer was dried over MgSO₄. The solvent wasdistilled off under reduced pressure, and then the residue was subjectedto silica gel column chromatography (SiO₂ 250 g, MeOH—CHCl₃ 3:97 v/v) toobtain(5R)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one(6.00 g, 98.8%).

[0151] (3) (5R)-5-(bromomethyl)-1-methylpyrrolidin-2-one

[0152] Bu₄NF (1 mol THF solution, 14.1 ml, 14.1 mmols) was added to aTHF (100 ml) solution of the(5R)-5-[(tert-butyldimethylsilyloxy)methyl]-1-methylpyrrolidin-2-one(3.43 g, 14.1 mmols), and the mixture was stirred for 15 hours at roomtemperature. The reaction mixture was concentrated under reducedpressure, and then the residue was subjected to silica gel columnchromatography (SiO₂ 150 g, MeOH—CHCl₃ 1:9 v/v) to obtain(5R)-5-(hydroxymethyl)-1-methylpyrrolidin-2-one as a mixture (2.48 g)with inseparable impurities.

[0153] Ph₃P (5.04 g, 19.2 mmols) was added to a CH CN (60 ml) solutionof the above mixture (2.48 g), and then a CH₃CN (15 ml) solution of CBr₄(6.37 g, 19.2 mmols) was added dropwise during cooling with ice,followed by stirring the mixture for 15 hours at room temperature. Thereaction mixture was concentrated under reduced pressure, and then theresidue was subjected to silica gel column chromatography (SiO₂ 150 g,AcOEt) to obtain (5R)-5-(bromomethyl)-1-methylpyrrolidin-2-one (2.68 g,98.5%).

[0154] (4) 2-((2R)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile

[0155] KCN (1.26 g, 25.7 mmols), NaCN (2.78 mg, 42.7 mmols) and18-crown-6 (793 mg, 3 mmols) were added to a CH₃CN (60 ml) solution ofthe (5R)-5-(bromomethyl)-1-methylpyrrolidin-2-one (2.67 g, 13.9 mmols),and the mixture was heated for 44 hours under reflux. The inorganicmatter was filtered off, and the filtrate was diluted with AcOEt (50ml). The dilution was washed with a saturated aqueous solution (30 ml)of NaCl, and dried over MgSO₄. The solvent was distilled off underreduced pressure, and then the residue was subjected to silica gelcolumn chromatography (SiO₂ 100 g, MeOH—CHCl₃ 1:9 v/v) to obtain2-((2R)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile as a mixture (1.49g) with impurities difficult to separate.

[0156] (5) 2-((2R)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile

[0157] Lawesson's reagent (Tokyo Kasei Kogyo) (2.26 g, 5.6 mmols) wasadded to a benzene (40 ml) solution of the2-((2R)-1-methyl-5-oxopyrrolidin-2-yl)ethanenitrile (1.48 g, 10.7mmols), and the mixture was heated for 2 hours under reflux. The solventwas distilled off under reduced pressure, and then the residue wassubjected to silica gel column chromatography (SiO₂ 80 g, MeOH—CHCl₃5:95 v/v) to obtain2-((2R)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile (1.36 g, 82.8%).

[0158] (6)N-[2-((2R)-1-methylpyrrolidin-2-yl)ethyl](4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoamide

[0159] Raney-Ni (1.5 ml) was added to an EtOH (30 ml) solution of the2-((2R)-1-methyl-5-thioxopyrrolidin-2-yl)ethanenitrile (1.36 g, 8.8mmols), and the mixture was heated for 24 hours under reflux (Upon TCL,the starting materials had not completely disappeared). The inorganicmatter was filtered off, and then the solvent was distilled off underreduced pressure. The residue was subjected to silica gel columnchromatography (SiO₂ 40 g, MeOH—CHCl₃ 1:9 v/v) to obtain2-((2R)-1-methylpyrrolidin-2-yl)ethanenitrile as a mixture (498 mg) withimpurities difficult to separate.

[0160] To a THF (15 ml) suspension of LiAlH₄ (130 mg, 3.4 mmols), a THF(5 ml) solution of the 2-((2R)-1-methylpyrrolidin-2-yl)ethanenitrilemixture (249 mg) was added dropwise little by little, with stirring,during cooling with ice. Then, the mixture was heated under reflux for45 minutes. After cooling, c. NH₄OH was added, and the reaction mixturewas filtered using Celite. The filtrate was distilled under reducedpressure to remove the solvent, whereafter CH₂Cl₂ was added to theresidue, and the mixture was dried over K₂CO₃. The solvent was distilledoff under reduced pressure. CH₂Cl₂ (12 ml) was added to the resultingcrude product, 2-((2R)-1-methylpyrrolidin-2-yl)ethylamine (257 mg), thena CH₂Cl₂ (3 ml) solution of DHA-Cl (350 mg, 1.01 mmols) was added, andthe mixture was stirred for 15 hours at room temperature. The reactionmixture was concentrated under reduced pressure, and then the residuewas subjected to silica gel column chromatography (SiO₂ 30 g, MeOH—CHCl₃1:9 v/v saturated with a concentrated aqueous solution of NH₄OH) toobtain the captioned compound (314 mg, 35.8%): [α]_(D) ²⁴+2.74 (c 0.786,CHCl₃)

Example 14 Production of an 0/W Type Emulsion Preparation of Compound 2(Hereinafter Referred to as Compound 2-MS)

[0161] An 0/W type emulsion preparation of Compound 2 (Compound 2-MS)was produced in the following manner: Purified egg yolk lecithin (6.0 g)and 50.0 mg of Compound 2 were added to 50.0 g of purified soybean oil,and these materials were melted with heating at 40 to 45° C. To themelt, 11.5 mg of glycerin (The Pharmacopoeia of Japan) was added, andthen distilled water for injection was used to give a total amount of500 ml. The resulting mixture was formed into a coarse emulsion by meansof a homomixer. The coarse emulsion was passed 5 times through aMenton-Gorin homogenizer at a pressure of 600 kg/cm², whereby it wasfinely emulsified. As a result, a homogenized, very fine Compound 2-MSpreparation was obtained. The mean particle size and the particle sizedistribution of the so produced Compound 2-MS preparation were measuredby Nicomp 370/Autodilute Submicron Particle Sizer (a product of PacificScientific). The mean particle size was 0.15 to 0.3 μm, and the particlesize distribution was such that no particles measuring 1 μm or more werecontained.

Test Example 1 In vitro Platelet Aggregation

[0162] Collagen-Induced In Vitro Platelet Aggregation

[0163] It was studied whether the compounds of the present inventionwould inhibit platelet aggregation induced by collagen.

[0164] Study Drugs:

[0165] The compounds of the Examples (Compounds 1 through 9 and 11) wereused in experiments. These compounds were each diluted withphysiological saline to end concentrations of 1×10⁻⁷M, 3×10⁻⁷M, 1×10⁻⁶M,3×10⁻⁶M, 1×10⁻⁵M, 3×10⁻⁵M and 1×10⁻⁴M.

[0166] Indomethacin was used as a positive control. Indomethacin wasdissolved in methanol, and then diluted with physiological saline to endconcentrations of 1×10⁻⁶M, 3×10⁻⁶M and 1×10⁻⁵M.

[0167] Physiological saline was used as a negative control.

[0168] Preparation of Platelets:

[0169] After a male JW rabbit (SPF, 2.5-month old) was anesthetized,blood was withdrawn from the carotid artery into a tube containing 3.8%sodium citrate (Citral, Yamanouchi Pharmaceutical). The tube wascentrifuged at 900 rpm for 10 minutes at room temperature to separatethe supernatant (PRP, platelet rich plasma fraction). The residue wasfurther centrifuged for 15 minutes at 2,500 rpm to obtain thesupernatant (PPP, platelet poor plasma fraction).

[0170] Measurement of Transmittance:

[0171] PPP (platelet poor plasma fraction) was placed in a cuvette, andthe cuvette was mounted in a 37° C. incubator for PPP of an aggregometer(Hematracer 240 12ch, manufactured by MCM). Then, 200 μl of PRP(platelet rich plasma fraction) was added into a cuvette, and thecuvette was mounted in a reactor for PRP. With stirring by a magneticstirrer, the transmittance of PPP was corrected to T_(650nm)=100%, andthe transmittance of PRP was corrected to T_(650nm)=0%, by automaticadjustment for 30 seconds.

[0172] The study substance (20 μl) at a predetermined concentration wasadded into the cuvette and, 30 seconds later, 20 μl (end concentration0.2 μg/ml) of collagen (produced by MCM) was added. Subsequent changesin the transmittance were recorded for 5 minutes. When the platelets areaggregated, the transmittance increases. When aggregation peaked, thetransmittance T at maximum aggregation was compared with thetransmittance T₀ of the negative control, and the aggregation inhibitionrate was expressed as percentage using the following equation:

Aggregation inhibition rate=(1−T/T ₀)×100

[0173] From an aggregation inhibition curve obtained, a regressionequation was derived by approximation of the linear type. IC₅₀, thepoint of 50% inhibition, was calculated from the regression equation.

[0174] The aggregation inhibition curve is shown in FIG. 1. IC₅₀, thepoint of 50% inhibition of platelet aggregation, is shown in thefollowing Table 1. TABLE 1 50% Inhibition Point for Platelet AggregationStudy drug IC₅₀ Indomethacin (5.0 ± 1.6) × 10⁻⁶ M Compound 1 (7.9 ± 1.3)× 10⁻⁶ M Compound 2 (1.5 ± 2.0) × 10⁻⁶ M Compound 3 (8.1 ± 3.3) × 10⁻⁶ MCompound 4   (42 ± 22) × 10⁻⁶ M Compound 5 (5.6 ± 2.8) × 10⁻⁶ M Compound6 (2.1 ± 0.1) × 10⁻⁶ M Compound 7 (3.7 ± 1.9) × 10⁻⁶ M Compound 8 (3.7 ±1.9) × 10⁻⁶ M Compound 9 (1.5 ± 0.9) × 10⁻⁶ M Compound 11 (4.2 ± 2.2) ×10⁻⁶ M

[0175] The above results show that Compounds 1 to 9 and 11 suppressedplatelet aggregation to practically the same degree as did indomethacin.

[0176] Thus, the compounds of the present invention represented by theformula I, where n in C_(n)H_((2n−2m)) is an integer of 6 to 20, and theunsaturation number m in C_(n)H_((2n−2m)) is an integer of 0 to 6, wereconfirmed to have a platelet aggregation suppressing action.

[0177] In Vitro Platelet Aggregation by Various Inducers

[0178] Next, the suppression of platelet aggregation by the compounds ofthe present invention was studied in the same manner as described abovewith the use of not only collagen, but also arachidonic acid, ADP,thrombin, serotonin, epinephrine and U46619 (pseudo-thromboxane A2substance) as inducers.

[0179] As platelets, platelets prepared from rats and guinea pigs aswell as rabbits were also used.

[0180] Preparation of aggregation inducers was performed in thefollowing manner:

[0181] Collagen and ADP were purchased from MCM, and used in accordancewith the method of preparation described in the manuals attached to thepurchased products. Serotonin, epinephrine and thrombin were purchasedfrom Sigma, and used as solutions in physiological saline for injection(Otsuka Pharmaceutical Factory). Arachidonic acid was purchased fromCayman, and dispersed in physiological saline for injection (OtsukaPharmaceutical Factory) with the use of an immersion type ultrasonicoscillator (US150, NIPPON SEIKI, tip end diameter ca. 3.5 mm). U46619(pseudo-thromboxane substance) was purchased from FUNAKOSHI, anddissolved in physiological saline for injection (Otsuka PharmaceuticalFactory) when used.

[0182] Collagen (0.1 to 1 μg/ml), arachidonic acid (1 mM), ADP (1 μM),thrombin (1 U/ml), serotonin (100 μM), epinephrine (100 to 200 μM), andU46619 (3 μM) were used.

[0183] Compound 2 was used as the study drug. Indomethacin was used as apositive control for measuring the suppression of platelet aggregationinduced by collagen.

[0184] IC₅., the point of 50% inhibition of platelet aggregation, wasdetermined, and is shown in the following Table 2. TABLE 2 50%inhibition point, IC₅₀, for in vitro platelet aggregation by variousinducers Inducer Rabbit Rat Guinea pig Collagen 2.1 ± 0.8 μM 11 ± 7 μM9.0 ± 6.0 μM Compound 2 (Indomethacin) (4.0 ± 0.5 μM) (18 ± 7 μM) (29 ±23 μM) ADP >100 μM >100 μM 65 ± 19 μM Thrombin N.A. — >100 μMArachidonic acid >100 μM N.A. — Epinephrine — — N.A. Serotonin — — N.A.U46619 50 ± 10 μM N.A.

[0185] In the case of collagen-induced aggregation, whether theplatelets from the rat or the guinea pig were used, the 50% aggregationinhibition point by Compound 2 was about (11±7)×10⁻⁶ M and (9.0±6.0)×10⁻⁶ M, respectively. These findings showed that Compound 2 exhibited aplatelet aggregation inhibiting action nearly comparable to that ofindomethacin, as in the case where rabbit's platelets were used.

[0186] In the case of aggregation induced by arachidonic acid, ADP andthrombin, whether the platelets from the rabbit, the rat or the guineapig were used, it was found that Compound 2 either did not inhibitplatelet aggregation, or even when it inhibited platelet aggregation,its degree of inhibition was very low.

[0187] The reason why data were not acquired in some of the experimentsmay have been that there were animal species differences in the activityof the platelets, and the sensitivity of the particular platelets to theparticular inducers was low.

[0188] The same results as described above were obtained in theexperiments using Compound 1.

[0189] Hence, the compounds of the present invention were shown to becapable of selectively suppressing platelet aggregation induced bycollagen.

[0190] Human In Vitro Platelet Aggregation

[0191] Preparation of Human Platelets:

[0192] From each of the large brachial veins of human volunteers, 10 mlof blood per person was drawn using an 18G syringe needle. The blood wasgently dropped into a 15 ml Falcon tube containing 1 ml of 3.8% sodiumcitrate (Citral, Yamanouchi Pharmaceutical). A stopper for the tube wasclosed, and slowly inverted for mixing. Then, the blood was centrifugedat 900 rpm for 10 minutes at room temperature to separate thesupernatant (platelet rich fraction, platelet rich plasma, PRP). Theresidue was further centrifuged for 15 minutes at 2,500 rpm to obtainthe supernatant (platelet poor fraction, platelet poor plasma, PPP). Theplatelet density of PRP was measured with a blood cell counter (Sysmex),and diluted with PPP to adjust the platelet density to 300,000platelets/μl.

[0193] Preparation of aggregation inducers was performed in the samemanner as described earlier.

[0194] For each experiment, the doses of the inducers were so set as toobtain best evaluation of the action of the study substance. That is,the concentration of each inducer at which aggregation would clearlydevelop in the presence of a negative control (physiological saline) butwould not excessive was set for each experiment.

[0195] If aggregation is induced by high concentration ADP or highconcentration epinephrine, on the other hand, primary aggregation takesplace, and then secondary aggregation proceeds again, under the action,as a trigger, of alpha granules (PDGF, serotonin, ADP, etc.) liberatedfrom activated platelets. In the present study, the effect of the studysubstance on secondary aggregation due to ADP or epinephrine was alsoinvestigated.

[0196] Serotonin, when used alone, minimally causes plateletaggregation. Thus, a combination of serotonin and collagen, or acombination of serotonin and ADP was used as the inducer. The dose ofthe inducer was set by examining, for each experiment, the dose of eachinducer which caused no aggregation when used alone, and combining doseslower than the doses which did not cause aggregation. For example, ifserotonin used alone induced aggregation at a dose of 1 mM, but inducedlittle aggregation at a dose of 0.5 mM, and if ADP alone inducedaggregation at a dose of 0.25 mM, but induced no aggregation at a doseof 0.11 mM, then the doses, 0.25 mM serotonin +0.05 mM ADP, were set.

[0197] Compound 2 was used as the study substance, and the preparationof the inducers was performed in the same manner as described earlier.

[0198] The measurement of the transmittance was carried out in the samemanner as described earlier.

[0199] IC₅₀, the point of 50% inhibition of platelet aggregation, wasdetermined, and is shown in the following Table 3. TABLE 3 50%inhibition point, IC₅₀, of Compound 2 against human in vitro plateletaggregation Inducer Concentration of inducer IC₅₀ (M) Serotonin +Collagen (0.25˜0.5) μM + (0.05˜0.2) μg/ml (1.5 ± 0.8) × 10⁻⁵ Serotonin +ADP (0.25˜1) μM + (0.05˜0.25) μM (3.0 ± 1.6) × 10⁻⁶ Collagen(0.075˜0.25) μg/ml (1.3 ± 0.2) × 10⁻⁵ ADP (primary) (0.5˜1) μM (3.0 ±1.9) × 10⁻⁵ (secondary) (1˜2.5) μM (5.7 ± 2.6) × 10⁻⁷ Epinephrine(primary) 1 μM (3.3 ± 0.6) × 10⁻⁵ (secondary) (1˜3) μM (2.7 ± 1.2) ×10⁻⁶ Arachidonic acid (1˜2) μM (8.0 ± 1.9) × 10⁻⁵ U46619(pseudo-thromboxane A2) (5˜10) μM (5.3 ± 2.3) × 10⁻⁵ Thrombin (0.2˜0.25)U/ml (5.7 ± 0.2) × 10⁻⁵

[0200] As shown in the table, whichever inducer caused aggregation, theaggregation suppressing action of Compound 2 was observed. Particularly,the suppressive action of Compound 2 against ADP-induced secondaryaggregation, epinephrine-induced secondary aggregation,serotonin-induced aggregation, or collagen-induced aggregation wasclearly observed. The values of the 50% suppression point, IC₅₀, againstthe respective inducers were as follows: (5.7±2.6)×10⁻⁷ M (ADP secondaryaggregation), (2.7±1.2)×10⁻⁶ M (epinephrine secondary aggregation),(3.0±1.6)×10⁻⁶ M (serotonin+ADP), (1.5±0.8)×10⁻⁵ M (serotonin+collagen),and (1.3±0.2)×10⁻⁵ M (collagen).

Test Example 2 Ex Vivo Platelet Aggregation

[0201] Study of the Onset of Pharmaceutical Efficacy

[0202] Compound 2 in a dose of 10 mg/kg was administered to the caudalvein of male SD rats (SPF, 8-week-old). The blood was withdrawn at threepoints in time, 10 minutes, 30 minutes and 1 hour after administration.Platelets were prepared from the blood samples taken, and they were usedto study the aggregation rates when platelet aggregation was induced by0.3 to 6 μg/ml of collagen. In a control group, the aggregation ratesafter administration of a solvent (physiological saline) were measured.The preparation of platelets and the measurement of the aggregationrates were performed in the same manner as in Test Example 1.

[0203] The results are shown in Table 2.

[0204] At any of the time points, 10 minutes, 30 minutes and 1 hourafter administration of Compound 2, platelet aggregation was suppressedin comparison with the solvent group. The suppressive action was foundto be such that platelet aggregation was suppressed more potently 1 hourafter intravenous administration than 10 minutes or 30 minutes afteradministration. Thus, the time point of measurement in subsequentexperiments was set to be 1 hour after administration.

[0205] The reason why platelet aggregation was suppressed more potently1 hour after intravenous administration than 10 or 30 minutes afterintravenous administration is assumed to be that this compound maygradually change into an active form in the blood, and its metabolitesmay contribute to its activity.

[0206] Study of Dose-Dependency

[0207] It was studied whether the compound of the present inventionwould suppress ex vivo platelet aggregation dose-dependently. Compound 2was used as the study substance, and a solvent (physiological saline)was used as a control.

[0208] Compound 2 in a dose of 1, 0.1, 0.01, 0.003, 0.001, 0.0003 or0.0001 mg/kg was administered to the rat caudal vein. The blood waswithdrawn 60 minutes after administration, and platelets were preparedfrom the blood. Platelet aggregation was induced by 4 to 7 μg/ml ofcollagen using the platelets. The preparation of platelets and themeasurement of the aggregation rate were performed in the same manner asin Test Example 1.

[0209] The results are shown in FIG. 3.

[0210] In the groups receiving 1, 0.1, 0.01 or 0.001 mg/kg of Compound2, platelet aggregation was suppressed in comparison with thesolvent-treated group. In the 0.0001 mg/kg group as well, plateletaggregation induced by collagen at a low concentration of 4 μg/ml wassuppressed compared with the solvent-treated group, although thesuppressive action was weak.

[0211] The value of the 50% suppression point, IC₅₀, was 630±36 ng/kgwhen calculated from the data obtained when platelet aggregation wasinduced by 4 μg/ml of collagen. This IC₅₀ value for suppression of exvivo platelet aggregation is about 1,000 times as high as that ofconventional GPIIb/IIIa receptor antagonists.

[0212] Separately, Compound 5, used as the study substance, wasintravenously administered in a dose of 0.1 mg/kg. The blood waswithdrawn 1 hour after intravenous administration, and platelets wereprepared from the blood. Platelet aggregation was induced by 1 to 6μg/ml of collagen using the platelets. The preparation of platelets andthe measurement of the aggregation rate were performed in the samemanner as in Test Example 1.

[0213] The results are shown in FIG. 4.

[0214] Compound 5 was also found to suppress platelet aggregation.

[0215] (Discussion)

[0216] The action of inhibiting ex vivo platelet aggregation, shown inthis Example, will be compared with the above-described action ofinhibiting in vitro platelet aggregation.

[0217] The IC₅₀ value of 630 ng/kg, obtained in the ex vivo experiments,can be presumed to be a dose at which the blood concentration ofCompound 2 reaches 26 nM, if the amount of rat blood is estimated atabout 60 ml/kg. This value is smaller than the IC₅₀ value of 2.1±0.8 μMin the in vitro experiments, and represents a low concentration which isabout 1/80 of the latter value.

[0218] That is, the action of inhibiting ex vivo platelet aggregationdoes not merely show that platelet aggregation is not simply suppressedby the compounds of the present invention. This action also suggests thepossibility, for example, that the compounds of the present inventionsuppress the interaction between neutrophils and platelets involved inplatelet aggregation, or that the compounds of the present invention aremetabolized, and platelet aggregation is strongly suppressed in vivowhile the metabolites is acting on platelets.

Test Example 3 Ex Vivo Platelet Aggregation After Oral Administration

[0219] Compound 2 in a dose of 10, 1 or 0.1 mg/kg was orallyadministered, as a single dose, to rats with the use of a probe needle.A solvent (physiological saline) was administered as a control.

[0220] The blood was withdrawn 2 hours after administration, andplatelets were prepared from the blood. Platelet aggregation was inducedby 2 to 4 μg/ml of collagen using the platelets. The preparation ofplatelets and the measurement of the aggregation rate were performed inthe same manner as in Test Example 1.

[0221] The results are shown in FIG. 5.

[0222] Platelet aggregation was potently suppressed in the groupsreceiving Compound 2, as compared with the solvent-treated group.

[0223] That is, the compounds of the present invention were shown to becapable of suppressing platelet aggregation even when absorbed orally.

Test Example 4 Collagen-Induced Mouse Sudden Death Model

[0224] As a preliminary study of the effect of the compounds of thepresent invention on an in vivo pathophysiological model, it wasinvestigated whether the compound of the present invention wouldsuppress collagen-induced mouse sudden death. In this model, pulmonarycapillaries are obstructed by a large amount of collagen, and a suddendeath is caused by oxygen deficiency. It was observed whether thecompound of the present invention would suppress platelet aggregation inthe body of the animal, thereby inhibiting a sudden death.

[0225] One hour after a solvent (physiological saline) or 30 μg/kg ofCompound 2 was intravenously administered to mice, 37.5 mg/kg ofcollagen was administered to the caudal vein. One hour later, thesurvival rate was compared between the groups. The following resultswere obtained:

[0226] Survival rate in the solvent group: 2/10 mice

[0227] Survival rate in the Compound 2 group: 7/10 mice

[0228] Compound 2 showed a higher survival rate than in the solventgroup.

Test Example 5 Rat Lauric Acid-Induced Peripheral Circulatory DisorderModel

[0229] (1) Administration After Induction of Leg Necrosis

[0230] A rat lauric acid-induced peripheral circulatory disorder model,which is widely used as a peripheral circulatory disorder model, wasused to investigate the effect of the compounds of the present inventionon an in vivo pathophysiological model of circulatory disorder.

[0231] Lauric acid (1.5 mg/animal) was administered into the rat femoralartery to induce leg necrosis. Six hours after lauric acidadministration, Compound 2 was intravenously administered as initialtreatment, and then repeatedly administered once daily for 14 days.

[0232] The single dose of Compound 2 was set at 10 μg/kg, 30 μg/kg or100 μg/kg to investigate whether this compound would ameliorate legnecrosis dose-dependently. A solvent (physiological saline) wasadministered as a control.

[0233] The progress of foot/leg lesions was observed 3, 7, 10 and 14days after lauric acid administration, and evaluated by the followingleg necrosis score. The score was given to each of the digits, and thesum of the scores of the respective digits was taken as a lesion index.If the disorder extended to the plantar portion, 5 points were furtheradded.

[0234] Score 0: No lesion.

[0235] Score 1: Blackening is limited to the tiptoe.

[0236] Score 2: Blackening extends to the digital portion.

[0237] Score 3: Necrosis of the digit.

[0238] Score 4: Loss of the digit.

[0239] The results are shown in FIG. 6. Compound 2 was shown to improvethe leg necrosis score dose-dependently.

[0240] (2) Administration Prior to Induction of Leg Necrosis

[0241] A test was conducted in the same manner as in (1) above, exceptthat the administration of Compound 2 was started 1 hour beforetreatment with lauric acid, then Compound 2 was administered 6 hoursafter administration of lauric acid, and Compound 2 was furtheradministered repeatedly once daily for 14 days. A solvent (physiologicalsaline) was similarly administered as a control.

[0242] The progress of foot/leg lesions was observed 3, 7, 10 and 14days after lauric acid administration, and evaluated by the above legnecrosis score.

[0243] The results are shown in FIG. 7. Compound 2 was demonstrated toimprove the leg necrosis score dose-dependently.

[0244] Upon prior administration, Compound 2 alleviated the disordersignificantly in doses of 30 and 100 μg/kg, and tended to alleviate thedisorder even in a dose of 10 μg/kg.

[0245] (3) Administration Prior to Induction of Leg Necrosis—O/WEmulsion Preparation

[0246] A test was conducted in the same manner as in (2) above.

[0247] However, Compound 2-MS (single dose: 100 μg/kg as the amount ofthe active ingredient) prepared in Example 14 was used as the studydrug, Palux (registered trademark) (Lipo PGE1, Taisho Pharmaceutical:single dose is 5 μg/kg as the amount of the active ingredient) orNovastan (registered trademark) (Argatroban, Mitsubishi-TokyoPharmaceuticals: single dose is 1 mg/kg as the amount of the activeingredient) was used as a positive control, and a solvent (physiologicalsaline) was used as a negative control.

[0248] The progress of foot/leg lesions was observed 3, 7, 10 and 14days after lauric acid administration, and evaluated by the above legnecrosis score.

[0249] The results are shown in FIG. 8. Compound 2-MS was shown to bemore potent than Argatroban and improve the leg necrosis score to adegree comparable to that of Lipo PGE1.

Test Example 6 Pseudo-Blood Vessel In Vitro Inflammation Model

[0250] A report says that when the inflammatory cytokine TNFα inducesadhesion molecules of vascular endothelial cells and causesinflammation, whereupon neutrophils migrate to the site of inflammation,aggravating inflammation. Severe inflammation destroys the homeostasisof circulatory organs, progressing arteriosclerosis. Thus, a study wasconducted on whether the compounds of the present invention have ananti-inflammatory action in a pseudo-blood vessel in vitro inflammationmodel using TNFα as an inflammation inducer.

[0251] Establishment of Pseudo-Blood Vessel in Vitro Inflammation Model

[0252] The pseudo-blood vessel in vitro inflammation model was preparedin accordance with the method described in “A Lecture onBiopharmaceutical Experiments, 12 Inflammation and Allergy II, pp.327-341” (edited by: Kazuo Ouchi, Hirokawa Publishing Company, publishedMay 15, 1993).

[0253] Transwells (KURABO INDUSTRIES), each separated into an upperchamber and a lower chamber by a porous polycarbonate membrane having 3μm pores, was used. A layer of bovine endothelial cells was adhered tothe bottom surface of the membrane, and cultured for 80 minutes at 37°C. in 5% CO₂. Then, a suspension of fluorescence-labeled neutrophils wasadded to the upper chamber of the transwell. Simultaneously, TNFα wassuspended in the cell suspension in the upper chamber to an endconcentration of 50, 25 or 17 ng/ml.

[0254] That is, in the above model, the phenomena that neutrophils passfrom the upper chamber through the vascular endothelial cell layer intothe lower chamber, and neutrophils adhere to the endothelial cell layermimic a state where in response to TNFα within the blood vessel,neutrophils migrate from within the blood vessel to the site ofinflammation.

[0255] The study showed that the number of neutrophils passing from theupper chamber through the vascular endothelial cell layer into the lowerchamber, and the number of neutrophils adhering to the endothelial celllayer increased in a manner dependent on the concentration of TNFα. Thatis, TNFα was demonstrated to cause inflammation.

[0256] Effect of the Compounds of the Present Invention on theInteraction Between Neutrophils and Vascular Endothelial Cells

[0257] Next, a study of how Compound 1 or Compound 2 would affect theinteraction between neutrophils and vascular endothelial cells wasconducted using the above pseudo-blood vessel in vitro inflammationmodel.

[0258] Compound 1 or Compound 2 was placed in the upper chamber of thetranswells to an end concentration of 30, 3 or 0.3 μM, together with asuspension of neutrophils and 50 ng/ml of TNFα. In the same manner asdescribed above, the number of neutrophils passing from the upperchamber through the vascular endothelial cell layer into the lowerchamber, and the number of neutrophils adhering to the endothelial celllayer were measured. Based on these numbers, the neutrophil migrationrate (%) was calculated. The neutrophil migration rate was expressed asthe relative value (%) of the number of the migrating neutrophils in thedrug treatment group with respect to the number of the migratingneutrophils in the negative control group (physiological saline).

[0259] The results are shown in FIG. 9.

[0260] Compound 1 and Compound 2 each suppressed neutrophil passagedose-dependently.

[0261] That is, the possibility was suggested that both of Compound 1and Compound 2 work suppressively toward the interaction betweenneutrophils and vascular endothelial cells, and act in the direction ofanti-inflammation.

[0262] Since Compound 1 or 2 acts in an anti-inflammatory manner,Compound 1 or 2 is expected to maintain homeostasis of circulatoryorgans and act in the direction of amelioration of pathological states.

Test Example 7 Photosensitization-Induced Rat Middle Cerebral ArteryObstruction Model

[0263] A photosensitization-induced rat middle cerebral arteryobstruction model, widely used as a cerebral infarction acutephase/chronic phase model, was used to investigate the effect of thecompounds of the present invention on an in vivo circulatory disorderpathophysiological model.

[0264] After rats were intravenously injected with a rose bengal dye (20mg/kg), the middle cerebral artery was irradiated with green laser light(wavelength 540 nm) for 10 minutes to induce arterial infarction due toactive oxygen species. After completion of irradiation with laser light,Compound 2 was intravenously administered.

[0265] The dose of Compound 2 was set at 1 mg/kg, and whether thiscompound would ameliorate cerebral infarction was investigated. Asolvent (physiological saline) was intravenously administered as acontrol.

[0266] The progress of cerebral infarction was observed 24 hours afterlaser light irradiation. A transverse section (1 mm in thickness) of thebrain exenterated was prepared, and soaked in triphenyltetrazolium tostain living tissue red. An infarct was white, and was clearlydistinguished from the living tissue. Thus, the area of the infarct wasmeasured, and the infarct rate (the proportion of the area of theinfarct to the transverse section of the brain) was calculated for usein evaluation.

[0267] The results are shown in FIG. 10. Compound 2 was shown toameliorate cerebral infarction.

Test Example 8 Rat Carotid Artery Tunica Intima Thickening Model

[0268] A rat carotid artery tunica intima thickening model, widely usedas a post-PTCA restenosis model, was used to investigate the effect ofthe compounds of the present invention on an in vivo circulatorydisorder pathophysiological model.

[0269] The tip of a balloon catheter (Fogarty catheter 2Fr, Baxter) wasinserted through the rat femoral artery, and led to the carotid artery.Air (0.3 ml) was injected into the balloon to inflate the balloon. Withthe balloon in an inflated state, the balloon catheter was pulled out upto the aortic arch. In this manner, the carotid artery tunica intima wasinjured 3 times. The O/W type emulsion preparation of Compound 2(Compound 2-MS) prepared in Example 14 was repeatedly administeredintravenously once daily, beginning 1 week before vascular tunica intimainjury, and repeatedly administered intravenously once daily for 2 weekseven after vascular tunica intima injury.

[0270] The single dose of Compound 2-MS was set at 100 μg/kg or 300μg/kg (as the amount of the active ingredient) to investigate whetherthis compound would suppress vascular tunica intima thickeningdose-dependently. An O/W type emulsion preparation, which does notcontain Compound 2, was administered as a negative control. Enalapril(Sigma) was used as a positive control, and administered in a dose of 30mg/kg once daily by the oral route in the same manner as describedabove.

[0271] Vascular tunica intima thickening was evaluated 2 weeks afterinjury. A transverse section of the carotid artery exenterated wasstained with Hematoxylin-Eosin (HE), and the area of the blood vessellumen, the area surrounded with the internal elastic lamina, and thearea surrounded with the external elastic lamina were measured. Tunicaintima thickening was evaluated by using the ratio of the area of theneogenetic tunica intima (the area surrounded by the internal elasticlamina −(minus) the area of the blood vessel lumen) to the area of thetunica media (the area surrounded with the external elastic lamina−(minus) the area surrounded by the internal elastic lamina).

[0272] The results are shown in FIG. 11. Compound 2-MS was clearly shownto suppress tunica intima thickening dose-dependently. The potency ofaction by 300 μg/kg of Compound 2-MS was nearly comparable to that of 30mg/kg of enalapril.

Test Example 9 Study of Synthetic Type Vascular Smooth Muscle CellProliferation

[0273] The effect of the study substance on vascular smooth muscle cellproliferation was studied.

[0274] It is speculated that with the progress of arteriosclerosis,vascular smooth muscle cells are transformed from the contractile typeinto the synthetic type, and while secreting inflammatory cytokines suchas PDGF, vascular smooth muscle cells are proliferated, resulting in theprogression of arteriosclerotic lesions (Roth's hypothesis).

[0275] Thus, the effect of Compound 2 on the cell proliferation ofvascular smooth muscle cells was measured in the following manner:

[0276] The rat carotid arterial tunica intima was rubbed by ballooning,and vascular smooth muscle cells were prepared by explant culture. Twoweeks later, these cells were cultured in a DMEM culture medium (Gibco)containing 10% fetal bovine serum. The cultures were subcultured severaltimes for stabilization, and then planted at a cell density of 5×10³cells/cm² for use in experiments. Compound 2 in combination with 50ng/ml of the growth factor PDGF (Sigma) was added to the above plantedcells and, 24 hours later, the cell density was measured by BrdU assay(Science '82, 218, p. 474, Cytometry '85, 6, p. 584). The cell densityin the absence of the drug was measured as a control.

[0277] Compound 2 was found to suppress vascular smooth muscle cellproliferation concentration-dependently at a concentration of 0.3 to 3μM. The results are shown in FIG. 12.

[0278] Relative smooth muscle cell count (%)=[cell count in theexperimental group]/[cell count in the control]×100

INDUSTRIAL APPLICABILITY

[0279] The compounds of the present invention can potently suppressplatelet aggregation (especially, platelet aggregation induced bycollagen), can suppress inflammation, and show an excellent prophylacticor therapeutic effect on circulatory diseases (for example, thromboticdiseases, arteriosclerotic diseases or hyperlipemic diseases).

1. (Deleted)
 2. (Amended) An aliphatic compound of the formula II, or astereoisomer thereof, or a pharmaceutically acceptable salt of saidaliphatic compound or said stereoisomer:

wherein A represents ch₃C_(n)H_((2n−2m))— wherein n denotes an integerof 4 to 22, and m represents an unsaturation number which is an integerof 0 to 7, l represents an integer of 0 to 10, R represents an alkylgroup having 1 to 10 carbon atoms which may be straight-chain orbranched-chain, and R^(A) represents hydrogen or an alkyl group having 1to 10 carbon atoms which may be straight-chain or branched-chain: 3.(Deleted)
 4. (Amended) The aliphatic compound, or the stereoisomerthereof, or the pharmaceutically acceptable salt of said aliphaticcompound or said stereoisomer, according to claim 2, wherein saidaliphatic compound is(4Z,7Z,10Z,13Z,16Z,19Z)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]docosahexaenoamide.5. (Deleted)
 6. (Amended) A method for producing the compound of claim2, comprising: reacting a compound of the formula VI′

wherein 1, R and R^(A) are as defined in claim 2, with a reactionproduct formed by a reaction between a compound of the formula A-CO—OH[wherein A represents CH₃C_(n)H_((2n−2m))— (wherein n and m are asdefined in claim 1)] and (CoCl)₂.
 7. (Amended) A pharmaceuticalcomposition for suppressing platelet aggregation, said pharmaceuticalcomposition comprising, as an active ingredient, the compound or thepharmaceutically acceptable salt thereof according to claim 2 or
 4. 8.(Amended) A pharmaceutical composition for preventing or treatingcirculatory disease, said pharmaceutical composition comprising, as anactive ingredient, the compound or the pharmaceutically acceptable saltthereof according to claim 2 or
 4. 9. (Amended) A pharmaceuticalcomposition for preventing or treating obstructive arteriosclerosis,said pharmaceutical composition comprising, as an active ingredient, thecompound or the pharmaceutically acceptable salt thereof according toclaim 2 or
 4. 10. (Amended) A pharmaceutical composition for suppressinginflammation, said pharmaceutical composition comprising, as an activeingredient, the compound or the pharmaceutically acceptable salt thereofaccording to claim 2 or
 4. 11. (Amended) A pharmaceutical compositionfor treating cerebral infarction, said pharmaceutical compositioncomprising, as an active ingredient, the compound or thepharmaceutically acceptable salt thereof according to claim 2 or
 4. 12.(Amended) A pharmaceutical composition for treating or preventingrestenosis after PTCA, said pharmaceutical composition comprising, as anactive ingredient, the compound or the pharmaceutically acceptable saltthereof according to claim 2 or
 4. 13. (Added) A pharmaceuticalcomposition for suppressing platelet aggregation, said pharmaceuticalcomposition comprising, as an active ingredient, an aliphatic compoundof the formula III, or a stereoisomer thereof, or a pharmaceuticallyacceptable salt of said aliphatic compound or said stereoisomer:

wherein A represents an optionally substituted CH₃C_(n)H_((2n−2m))—wherein n denotes an integer of 4 to 22, and m represents anunsaturation number which is an integer of 0 to 7, l represents aninteger of 0 to 10, R represents an alkyl group having 1 to 10 carbonatoms which may be straight-chain or branched-chain, and R^(A)represents hydrogen or an alkyl group having 1 to 10 carbon atoms whichmay be straight-chain or branched-chain.
 14. (Added) The pharmaceuticalcomposition according to claim 13, wherein said active ingredient is(4Z,7Z,10Z,13Z,16Z,19Z)—N-(4-methylpiperazin-1-yl)docosahexaenoamide, oran optical isomer thereof, or a pharmaceutically acceptable salt of saiddocosahexaenoamide or said optical isomer.
 15. (Added) A pharmaceuticalcomposition for suppressing inflammation, said pharmaceuticalcomposition comprising, as an active ingredient, an aliphatic compoundof the formula III, or a stereoisomer thereof, or a pharmaceuticallyacceptable salt of said aliphatic compound or said stereoisomer:

wherein A represents an optionally substituted CH₃C_(n)H_((2n−2m))—wherein n denotes an integer of 4 to 22, and m represents anunsaturation number which is an integer of 0 to 7, l represents aninteger of 0 to 10, R represents an alkyl group having 1 to 10 carbonatoms which may be straight-chain or branched-chain, and R^(A)represents hydrogen or an alkyl group having 1 to 10 carbon atoms whichmay be straight-chain or branched-chain.
 16. (Added) The pharmaceuticalcomposition according to claim 15, wherein said active ingredient is(4Z,7Z,10Z,13Z,16Z,19Z)-N-(4-methylpiperazin-1-yl) docosahexaenoamide,or an optical isomer thereof, or a pharmaceutically acceptable salt ofsaid docosahexaenoamide or said optical isomer.
 17. (Added) Apharmaceutical composition for preventing or treating circulatorydisease, said pharmaceutical composition comprising an aliphaticcompound of the formula III, or a stereoisomer thereof, or apharmaceutically acceptable salt of said aliphatic compound or saidstereoisomer:

wherein A represents an optionally substituted CH₃C_(n)H_((2n−2m))—wherein n denotes an integer of 4 to 22, and m represents anunsaturation number which is an integer of 0 to 7, l represents aninteger of 0 to 10, R represents an alkyl group having 1 to 10 carbonatoms which may be straight-chain or branched-chain, and R^(A)represents hydrogen or an alkyl group having 1 to 10 carbon atoms whichmay be straight-chain or branched-chain.
 18. (Added) The pharmaceuticalcomposition according to claim 17, wherein said circulatory disease isobstructive arteriosclerosis.
 19. (Added) The pharmaceutical compositionaccording to claim 17, wherein said circulatory disease is cerebralinfarction.
 20. (Added) The pharmaceutical composition according toclaim 17, wherein said circulatory disease is restenosis after PTCA. 21.(Added) The pharmaceutical composition according to any one of claims 17to 20, wherein said active ingredient is(4Z,7Z,10Z,13Z,16Z,19Z)-N-(4-methylpiperazin-1-yl) docosahexaenoamide,or an optical isomer thereof, or a pharmaceutically acceptable salt ofsaid docosahexaenoamide or said optical isomer.